Growing world shortages of food and feed, as well as an expanding world population has expanded the need for new, improved and increased food sources. Efforts are being directed to increasing the protein content of foods as well as increasing the available protein contained within those foods.
Referring to rapeseed and canola as an example, the harvested bean or seed typically has about 8% or more moisture content. Several extraction methods, including mechanical (pressing), solvent, enzymes and supercritical CO2 extraction, are used to remove the oil which is typically about 33% to about 50% of the harvested bean. Pressing is a simple process and has a low capital investment cost but suffers from high operating cost for power, significant equipment wear and tear, and labor costs. In addition, temperature increases due to mechanical working of the bean can result in quality degradation of the oil and meal. There is also a significant amount of residual oil left in the meal (about 15% to about 25%).
Solvent extraction is a more efficient method of oil removal. Hexane has had extensive use in the edible oil processing industry to remove the oil from the soy bean or canola seed. However, the rate of extraction is dependent on the meal flake surface area, temperature, the solvent selected and the moisture content of the seed (or bean). Hexane is typically applied to the pre-pressed seed but may also be applied after pressing of the cake. The extracted meal is then heated to drive off the hexane. The use of the solvent and heating tends to denature at least some of the protein rendering it insoluble. Also, the extracted meal still has from about 1 to about 4% by weight oil and may still have some residual hexane.
The oil is then separated from the solvent by heating the solution or steam injection. This procedure again suffers from oil quality degradation as a result of the use of elevated temperatures. Also, for food use, residual solvent remaining in the oil is a significant negative factor, requiring additional processing at elevated temperatures, which in turn further degrades the quality of the oil. For edible use, the oil must also be degummed to reduce phosphorus content. The desired product, referred to as super degummed oil, has between 10 and 30 ppm phosphorus. Crude degummed oil contains about 200 ppm as well as 0.5%–3.0% impurities of which about 2% is lecithin gums which are removed by water addition and separation. If not removed, these gums will settle out during storage, causing future processing problems.
Fatty acids are typically removed from the crude oil by saponification using sodium hydroxide to form a water soluble soap. The oil is then refined to lighten its color by removing beta-carotene and other colored constituents and deodorized using absorbents such as Fullers earth, clays, bleaching agents, steam stripping and distillation.
Supercritical CO2 extraction requires the application of high pressures (80–100 bar) to CO2 which is held above its critical temperature (31° C.) to maintain the carbon dioxide in a supercritical state. The seed is typically ground prior to exposure to the high pressure CO2. The meal may be digested using enzymes to leave the oil behind. High pressure, super critical processing produces a fluid CO2 extractant, which, because of the supercritical operating temperature and pressure, behaves like a liquid. The oil is released by lowering the pressure, which reduces the solubilization ability of the now gaseous CO2. A significant deficiency of this process is the capital investment in high pressure processing and CO2 recovery equipment.
Although the literature indicates many sources of plant proteins including starchy cereals (wheat, corn, oats, rye, barley, triticale, etc.), nuts, starchy legumes or lentils (field peas, chickpeas, fababeans, navy beans, pinto beans, etc.) and oilseeds (sunflower seed, rapeseed, canola, soy beans, peanuts, etc.), in general, the main source of commercial protein is the soybean. Industrial proteins separated from the source meal, normally called isolates, have protein contents of at least 90% expressed on a moisture free basis.
One process for preparing a soy protein isolate was described by Anson and Pader in 1957 (U.S. Pat. No. 2,785,155). The proteins in soy meal were dissolved using alkaline solubilization (high pH treatment), the insoluble material was removed by centrifugation and hydrochloric acid was added to the supernatant which contained the alkali-solubilized proteins. The acid precipitated the proteins isoelectrically thereby producing a highly proteinaceous product, i.e., a protein isolate. The isoelectric precipitation of soy proteins proved to be an economical and industrially practical method which has been the dominant technical approach for the preparation of protein isolates for a considerable period of time.
Sair (1959) in U.S. Pat. No. 2,881,076 disclosed an improved soy isolate of high yield, although the process still used an isoelectric precipitation step. Kraskin (1972) in Canadian Pat. No. 915,105 described an improved method for extracting proteinaceous materials using enzymes in addition to alkaline pH manipulation to solubilize maximal amounts of proteins; once this was achieved, precipitation of the proteins was done isoelectrically. Another improved process using slightly elevated temperature and pH manipulation to achieve high protein solubility was described by Calvert et al. (1973) in Canadian Pat. No. 917,995; the protein was then precipitated isoelectrically to yield a white, bland, homogeneous product. Boyer (1973) in Canadian Pat. No. 935,024 described a soy protein cheese-like curd which was prepared from isoelectrically precipitated protein; a heat step before precipitation produced a fluffy type of curd. A combination of heat and enzyme treatments was used by Hawley (1973) in Canadian Pat. No. 936,408 to produce a special protein preparation for acidic beverages and baking applications; once again the specially treated proteins were precipitated isoelectrically.
In U.S. Pat. No. 3,758,452 (Owen (1973)) a de-toxified rapeseed protein is prepared from press cake solubilized with sodium chloride. After particulate matter had been removed, the salt soluble protein was precipitated isoelectrically by the addition of acid. In a further example of this type of technology, Flink and Christiansen (The Production of a Protein Isolate from Vicia faba Lebesm.-Wiss. u Technol. 6: 102–106 (1973)), describe the preparation of a fababean (Vicia faba) protein isolate by protein solubilization at pH 8 to 10 and then isoelectric precipitation of the protein.
In the prior discussed procedures, the proteins are solubilized by alkaline or salt extraction with the treatment in some cases being enhanced by increased temperature, enzyme activity and/or salt addition. However, regardless of the solubilization scheme, isoelectric acid precipitation is used to separate the desired product. Furthermore, in order to efficiently produce a reasonable level of solubilized protein an alkaline pH step is normally required. However, there are some concerns regarding nutritive value of protein isolates prepared by alkaline solubilization and acid precipitation. deGroot and Slump (Effects of Severe Alkali Treatment of Proteins on Amino Acid Composition and Nutritive Value, Journal of Nutrition, 98: 45–56 (1969)), reported that alkali treated soy protein isolate contained the amino acid derivative lysinoalanine (LAL) which was absorbed poorly in the gut of growing animals. In fact, there was a negative correlation between LAL level in the diet and net protein utilization (N.P.U.) values. Then Woodard and Short, Toxicity of Alkali-Treated Soy Protein in Rats, Journal of Nutrition 103: 569–574(1973), confirmed the presence of LAL in alkali treated soy protein and showed an apparent correlation between LAL level and nephrotoxic reactions in rats. The common decrease in the protein efficiency ratio (P.E.R.) of soy isolates, when compared to soy flour and concentrate, is probably due to the formation of LAL on akali/acid processing and hence reduction of the essential amino acid lysine. Sternberg et al.(Lysinoalanine: Presence in Foods and Food Ingredients, Science, 190: 992–994(1975)), who found high levels of LAL in certain samples of sodium caseinate, dried egg white solids, and various processed foods as well as in foods heated under non-alkaline conditions. Additional concern over LAL in food systems has been generated by Gross (The Chemistry and Biology of Amino Acids in Foods Proteins, Agrochemistry Abstract #32 (1975), First Chemical Congress of the North American Continent, Mexico City), who showed that LAL can also cause the reabsorption of a developing foetus in the uteri of rats and rabbits. Therefore, there is a definite need for preparing protein isolates without alkali and heat treatments to reduce the generation of questionable amino acid derivative.
U.S. Pat. No. 4,169,090 describes a process for forming protein isolates which comprises subjecting a protein source material to a salt solution. The protein source material is first comminuted to provide an average particle size of between about 10 and about 800 mesh, usually less than about 200 mesh. This is followed by cell disruption and the physical removal of some non-proteinaceous material by screening, grinding, milling, air classifying, etc. The protein fraction (usually a dry flour or concentrate) is then mixed into a solution containing only water and an appropriate food grade salt (sodium chloride, potassium chloride, calcium chloride, etc. which has a normal pH of from about 5.0 to about 6.8) with agitation at 15° to 35° C. The insoluble particulate matter (usually cellular debris and perhaps starch granules) is removed from the solubilized proteins by settling, filtering, screening, decanting or centrifuging. The salt concentration is usually in the range of 0.2 to 0.8 ionic strength depending on the particular protein, the level of salts in that source material, the particle size of that material, the specific salt used and the extraction temperature and time. The resulting extract, referred to as a high-salt protein, contains many solubilized compounds in addition to proteins. The protein solution, usually has a protein concentration of about 10 to about 100 g/l. The protein solution is then concentrated using selective membrane technique.
The ionic strength of the solution of the solubilized proteins is then reduced. Various methods can be used such as membrane separation techniques (e.g., dialysis, ultra filtration, reverse osmosis, etc.) or dilution of the high salt protein extract by addition of water. Ionic strength reduction causes the protein structures formed by the addition of salt to dissociate causing a rapid decrease in molecular weight of the protein aggregates and the generation of a comparatively low molecular weight species. The resultant particles are small microscopic spheres containing many associated globular protein molecules called “protein micelles”.
In accordance with the U.S. Pat. No. 4,208,323, the yield of protein isolate which may be obtained by the prior process is increased by increasing the protein concentration of the protein solution in the extraction step while the salt concentration remains the same prior to the dilution step. Adding a washing step to the concentration steps significantly decreases the phosphorus concentration of the isolate.
U.S. Pat. No. 5,844,086 provides a further modification of the process of preparing a protein isolate starting from an oil seed meal having a fat content up to about 10 wt % of the meal. After addition of the salt solution the fat is removed from the protein solution to provide a defatted protein solution. The end product is a dried proteinaceous powder substantially undenatured and having a protein content of at least about 90 wt %.
U.S. Pat. No. 6,005,076 further improves the process to provide a purified protein isolate of high protein content from fat-contaminated oil seed meal. Centrifugation is used to reduce the moisture content of the protein solution from 70%–95% by weight to about 50%–80% by weight of the total isolate mass. This also decreases the occluded salt content of the isolate, and hence the salt content of dried isolate.
Applicant's published application WO 00/43471 is directed to a process for the extraction of fixed oils from various materials, such as seeds, using 1,1,1,2-tetrafluoroethane (HFC 134a) alone or in combination with co-solvents, at temperatures of 40° C. or above. Co-solvents include hydrocarbons, esters, ketones, chlorinated and fluorinated hydrocarbons, ethers, alcohols, etc. The oil is caused to separate from the solvent by lowering the temperature to room temperature or below, preferably 0–20° C. so the oil is no longer soluble and separates therefrom. A second of applicant's published applications, WO 01/10527, is directed to the extraction of fixed oils, fatty acids sterols, esters, natural waxes, hydrocarbons, flavors and fragrance oils and pigments from natural plant materials using iodotrifluoromethane (CIF3, also referred to herein as ITFM) alone or with cosolvents, such as HFC-134 at ambient temperature or below and slightly elevated pressures sufficient to keep the solvent in a liquid state. The oils are then separated out by reducing the pressure sufficient to cause the solvent to evaporate.